Chapter Six

The Behavior of Proteins:

Enzymes
Enzyme Catalysis
• Enzyme: a biological catalyst
• with the exception of some RNAs that
catalyze their own splicing (Section 10.4), all
enzymes are proteins
• enzymes can increase the rate of a reaction
by a factor of up to 1020 over an uncatalyzed
reaction
• some enzymes are so specific that they
catalyze the reaction of only one
stereoisomer; others catalyze a family of
similar reactions
• The rate of a reaction depends on its
activation energy, G°‡
• an enzyme provides an alternative pathway
with a lower activation energy
Enzyme Catalysis (Cont’d)

• For a reaction taking place at constant temperature

and pressure, e.g., in the body
A B
• the change in free energy is
G° = H° - T S°
• Difference in energies between initial state and final
state
G° = RT ln K eq

• The change in free energy is related to the

equilibrium constant, Keq, for the reaction by
Enzyme Catalysis (Cont’d)

• Consider the reaction

H2 O 2 H2 O + O2
Temperature dependence of catalysis

• Temperature can also

catalyze reaction (increase
rate)

• This is dangerous, why?

• Increasing temperature will

eventually lead to protein
denaturation
Enzyme Kinetics
• For the reaction
A + B P
• The rate of reaction is given by rate equation

[A] [B]
Rate = _ = _ = [P]
t t t
Rate = k[A] f[B] g

• Where k is a proportionality constant called the

specific rate constant
• Order of reaction: the sum of the exponents in the
rate equation
Enzyme Kinetics (Cont’d)
• Consider the reaction
A + B C + D
Whose rate equation is given by the expression
Rate = k[A] 1[B]1

• Determined experimentally, not always from balanced

equations
• The reaction is said to be first order in A, first order in
B, and second order overall
• Consider this reaction of glycogen with phosphate
Glycogen n + HPO 42- Glucose-1-phosphate + Glycogen n-1

Rate = k[Glycogen] 1[HPO 42-]1 = k[Glycogen][HPO 2-

4 ]
How Enzymes bind to Substrate

• In an enzyme-catalyzed reaction
• Substrate, S: a reactant
• Active site: the small portion of the enzyme surface
where the substrate(s) becomes bound by noncovalent
forces, e.g., hydrogen bonding, electrostatic
attractions, van der Waals attractions

E + S ES
enzyme-substrate
complex
Binding Models

• Two models have been developed to describe

formation of the enzyme-substrate complex

• Lock-and-key model: substrate binds to that portion

of the enzyme with a complementary shape

• Induced fit model: binding of the substrate induces a

change in the conformation of the enzyme that results
in a complementary fit
2 Modes of E-S Complex Formation
Formation of Product
An Example of Enzyme Catalysis

• Chymotrypsin catalyzes
• The selective hydrolysis of peptide bonds where the
carboxyl is contributed by Phe and Tyr

• It also catalyzes hydrolysis of the ester bonds

An Example of Enzyme Catalysis (Cont’d)
Non-Allosteric Enzyme Behavior
• Point at which the rate of
reaction does not change,
enzyme is saturated,
maximum rate of reaction
is reached
ATCase: An Example of Allosteric Behavior

• Sigmoidal shape- characteristic of allosterism

• Again Max. velocity reached, but different mechanism
Michaelis-Menten Kinetics

• Initial rate of an enzyme-catalyzed reaction versus

substrate concentration
Michaelis-Menten Model

• For an enzyme-catalyzed reaction

k1 k2
E + S ES P
k-1

• The rates of formation and breakdown of ES are

given by these equations

rate of formation of ES = k 1 [E][S]

rate of breakdown of ES = k -1 [ES] + k 2[ES]

• At the steady state

k1 [E][S] = k -1[ES] + k 2[ES]
Michaelis-Menten Model (Cont’d)

• When the steady state is reached, the concentration

of free enzyme is the total less that bound in ES

[E] = [E] T - [ES]

• Substituting for the concentration of free enzyme and

collecting all rate constants in one term gives
([E]T - [ES]) [S] k -1 + k 2
= = KM
[ES] k1

• Where KM is called the Michaelis constant

Michaelis-Menten Model (Cont’d)
• It is now possible to solve for the concentration of the
enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
= KM
[ES]
[E]T [S] - [ES][S] = KM [ES]

[E]T [S] = [ES](K M + [S])

• Or alternatively [E]T [S]

[ES] =
KM + [S]
Michaelis-Menten Model (Cont’d)
• In the initial stages, formation of product depends only on the
rate of breakdown of ES

k 2[E]T [S]
V init = k 2 [ES] =
KM + [S]
• If substrate concentration is so large that the enzyme is
saturated with substrate [ES] = [E]T

V init = V max = k 2 [E]T

• Substituting k2[E]T = Vmax into the top equation gives

Vmax [S] Michaelis-Menten

Vinit =
KM + [S] equation
Michaelis-Menten Model (Cont’d)
• When [S]= KM, the equation reduces to

V max [S] Vmax [S] Vmax

V= = =
KM + [S] [S] + [S] 2
Linearizing The Michaelis-Menten Equation
• It is difficult to determine Vmax experimentally
• The equation for a hyperbola

Vmax [S]
V= (an equation for a hyperbola)
KM + [S]
• Can be transformed into the equation for a straight line by taking
the reciprocal of each side

1 KM + [S] KM [S]
= = +
V V max [S] V max [S] V max [S]

1 KM 1
= +
V V max [S] V max
Lineweaver-Burk Plot
• The Lineweaver-Burke plot has the form y = mx + b, and is the
formula for a straight line
1 KM 1 1
= • +
V V max [S] V max
y = m • x + b
• a plot of 1/V versus 1/[S] will give a straight line with slope of
KM/Vmax and y intercept of 1/Vmax
• such a plot is known as a Lineweaver-Burk double reciprocal
plot
Lineweaver-Burk Plot (Cont’d)
• KM is the dissociation constant for ES; the greater the value of
KM, the less tightly S is bound to E

• Vmax is the turnover number

Turnover Numbers
• Vmax is related to the turnover number of enzyme:also
called kcat
V max 
  turnover _ number  kcat
[ET ] 
• Number of moles of substrate that react to form product
per mole of enzyme per unit of time
Enzyme Inhibition
• Reversible inhibitor: a substance that binds to an enzyme to inhibit it, but
can be released
• competitive inhibitor: binds to the active (catalytic) site and
blocks access to it by substrate
• noncompetitive inhibitor: binds to a site other than the active site;
inhibits the enzyme by changing its conformation

• Irreversible inhibitor: a substance that causes inhibition that cannot be

reversed
• usually involves formation or breaking of covalent bonds to or on
the enzyme
Competitive Inhibition
• Substrate must compete with inhibitor for the active
site; more substrate is required to reach a given
reaction velocity

EI I + E + S ES P

• We can write a dissociation constant, KI for EI

[E][I]
EI I + E KI =
[EI]
Competitive Inhibition
Competitive Inhibition
No inhibition
1 = KM • 1 + 1
V Vmax S Vmax
y = m • x + b

In the presence of a competitive inhibitor

1 KM [I] 1 1
= 1 + +
V Vmax KI S Vmax

y = m • x + b

• In a Lineweaver-Burk double reciprocal plot of 1/V

versus 1/[S], the slope (and the x intercept) changes
but the y intercept does not change
A Lineweaver-Burke Plot for Competitive
Inhibition
Noncompetitive Inhibition (Cont’d)

• Several equilibria are involved

+S
E ES E + P
-S
-I +I -I +I

+S
EI ESI
-S

• The maximum velocity Vmax has the form

V max
I
V max =
1 + [I]/K I
Noncompetitive Inhibition (Cont’d)
A Lineweaver-Burke Plot for
Noncompetitive Inhibition
• Because the inhibitor does not interfere with binding of substrate
to the active site, KM is unchanged
• Increasing substrate concentration cannot overcome
noncompetitive inhibition

No inhibition
1 = KM • 1 + 1
V Vmax S Vmax
y = m • x + b
In the presence of a noncompetitive inhibitor
1 KM [I] 1 1 [I]
= 1 + + 1 +
V Vmax KI S Vmax KI

y = m • x + b
A Lineweaver-Burke Plot for
Noncompetitive Inhibition (Cont’d)
Other Types of Inhibition

• Uncompetitive- inhibitor can bind to the ES complex

but not to free E. Vmax decreases and KM decreases.

• Mixed- Similar to noncompetitively, but binding of I

affects binding of S and vice versa.